The multicatalytic proteinase complex (MPC), also referred to as the proteasome, is a high molecular mass (approximately 700 kDa) intracellular particle composed of 28 to 32 low molecular mass subunits (22 to 34 kDa) of which 13 are nonidentical. The importance of the MPC for fundamental cell functions is indicated by the finding that it is present in all eukaryotic cells, that it is highly conserved in evolution, that it is involved in antigen processing, that it constitutes the 'proteolytic core' of the ubiquitin-dependent pathway for intracellular proteolysls and that it is indispensable for cell survival and proliferation. Changes in the expression of the MPC have been implicated in the pathophysiology of malignancies, and autoantibodies against the MPC have been found in serum of patients with systemic lupus erythematosus. The MPC exhibits three distinct proteolytic activities cleaving peptide bonds on the carboxyl side of basic, acid and hydrophobic amino acid residues, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine protease inhibitor. We have recently identified a fourth, DCI resistant component that cleaves peptide bonds on the carboxyl side of branched chain amino acids, and constitutes a major factor in the degradation of proteins and natural peptides. Major goals of this laboratory are to determine which and how many of the subunits of the MPC are proteolytically active, to identify their specificity and proteolytic mechanism, to examine factors that regulate the proteolytic activity and to synthesize specific inhibitors that could be used as probes in studies on the role of the complex in cellular functions. The aims of this proposal are: 1) to evaluate the specificity and regulation of activity of the fourth, DCI resistant component, 2) to determine if a recently identified prolyl aminopeptidase-like activity is expressed by a novel component of the MPG, and if so to investigate its properties and specificity, 3) to determine if a recently identified prolyl- endopeptidase-like activity is due to a unique component, and if so to examine its properties and specificity, 4) to investigate the structural requirements and kinetics of a group of peptides that activate the MPC, 5) to examine the function of the MPC in intact cells by studying the effect of specific inhibitors on intracellular protein degradation and on cell proliferation.